Figure 4: Polymerase complementation assay.

(a) Cartoon depicting the various PB2 mutants and the expected polymerase activity in the presence of these mutants. AvianPr-PB2-E627K-Tdef represents the AvianPr PB2 harbouring the mutation E627K and two mutations (E361A, F404A) in the cap-binding domain. Black solid arrows refer to synthesis of high RNA levels by the indicated polymerase; arrows with dotted lines indicate synthesis of low vRNA amounts. The question mark indicates unknown levels of RNA synthesis. (b) Determination of the mRNA, cRNA and vRNA levels by primer extension analysis after reconstitution of the AvianPr with the indicated PB2 subunits using specific primers for segment 6. Determination of the 5sRNA levels served as an internal loading control. For reconstitution the total amount of 50 ng of PB2-expressing plasmids were transfected. The complementation assays included 25 ng of AvianPr-PB2 and 25 ng AvianPr-PB2-E627K-Tdef. Omission of PB2 (−PB2) was used as a negative control. (c) Luciferase reporter activities of mutant polymerases 24 h post-transfection after reconstitution of the indicated polymerases in HEK293T cells. For each reconstitution, a total amount of 50 ng of PB2-expressing plasmids were used. The complementation assays included 25 ng of AvianPr-PB2 and 25 ng AvianPr-PB2-E627K-Tdef. Error bars indicate the standard error of the mean of three independent experiments. Student's t-test was performed to determine the P value. *P<0.05; **P<0.01. (d) Primary viral transcription in HEK293T cells infected with an MOI of 5 of either AvianPr or AvianPr-PB2-E627K and treated with 100 μg ml⁻¹ cycloheximide. Cells were collected 4 h p. i., and mRNA, cRNA and vRNA levels were determined using primer extension analysis with primers specific for segment 6. Levels of cellular 5sRNA served as internal loading control. (e) Rescue of cycloheximide-mediated inhibition of influenza virus replication. Human 293T cells were transiently transfected with the indicated polymerase subunits and viral NP and subsequently infected with the indicated virus strain at an MOI of 5. vRNA species were analysed 6 h post infection by primer extension using specific primer for segment 1.